PROJECT SUMMARY The overall aim of this proposal is to develop a toolkit designed to facilitate the functional annotation of human genes using Drosophila genetic studies. Numerous evolutionarily conserved genes can be studied in flies using a simple strategy. First, one inserts a T2A-GAL4-polyA artificial exon in the gene of interest. This can be done by converting the insertion sites of MiMIC transposable elements with T2A-GAL4-polyA or by direct integration of this cassette using CRISPR. These insertions typically create strong loss of function mutations. Moreover, the GAL4 transactivator is expressed in the same tissue and at the same time as the gene of interest. This can then be used to test if the fly or the homologous human cDNA is able to rescue the phenotype associated with the loss of the fly gene. If the human cDNA rescues, one has established that the two genes are orthologous. One can then determine the effect of human variants of interest (point mutations and polymorphisms) for functionality in flies, an approach that has already been shown to be extremely valuable for studies of human disease. To perform these experiments systematically we need to produce a library of human cDNAs that can be expressed in flies. We plan to create a resource for expressing ~8,000 epitope tagged human cDNAs of genes that are conserved between human and Drosophila. These cDNAs under the control of the UAS-GAL4 system will be inserted into a specific locus using the ?C31 integrase. We will make this library available to researchers via the Drosophila Genomics Resource Center. In addition, we will produce transgenic flies from a subset of these (1,500), using the following criteria: genes associated with known human diseases, genes with fly homologs that can be easily manipulated with available tools, and genes prioritized by other researchers (Drosophila biologists and human geneticists). These experiments will allow Drosophila and human researchers to test the functional replacement of genes for which some information is already available. The corresponding stocks will be deposited in the Bloomington Drosophila Stock Center. Finally, we will use the T2A-GAL4-polyA strategy to create strong loss of function mutations for about 500 Drosophila genes for which a MiMIC insertion is available. We will assess the phenotypes of a subset of these and test if the human cDNA is able to rescue the observed phenotypes in about 20 cases. This will establish how well the strategy works and provide valuable data for the community. Our goal is to provide molecular, genetic and transgenic resources for fly and human geneticists to accelerate the identification of human gene functions.